Synthesis of azo bonded immunoregulatory compounds

ABSTRACT

Methods are disclosed for preparing compounds of Formula I: 
     
       
         
         
             
             
         
       
     
     where R 1 , R 3 , and R 4  are independently hydrogen or C 1  to C 4  alkyl, and R 2  is: 
     
       
         
         
             
             
         
       
     
     where R 5  is selected from the group consisting of hydrogen and C 1  to C 4  alkyl, or 
     
       
         
         
             
             
         
       
     
     where R 6 , R 7  and R 8  are independently hydrogen or C 1  to C 4  alkyl; or the esters or pharmacologically acceptable salts thereof. The methods can involve converting a suitably functionalized aniline compound to a diazonium salt (which aniline compound can be first formed by reduction of a nitrobenzene) and coupling the diazonium salt with a suitably functionalized benzene compound. The suitably functionalized aniline compound either includes a primary alcohol or aldehyde group, which is then oxidized to a carboxylic acid group, or includes a nitrile or amide group, which is hydrolyzed to a carboxylic acid group. The methods can also involve the direct coupling (via reduction of nitro groups to form an azo linkage) of suitably functionalized nitrobenzenes. The compounds and or their metabolites can be used to treat or prevent various diseases, particularly inflammatory conditions of the GI tract.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/631,582, filed on Jan. 4, 2007, now U.S. Pat. No. 7,932,366,which in turn was filed under the provisions of 35 U.S.C. §371 andclaimed priority of International Patent Application No.PCT/US2005/024109, filed on Jul. 7, 2005, and which in turn claimspriority of US. Provisional Application No. 60/585,995 filed on Jul. 7,2004.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to immunoregulatory compounds, methods ofproducing same and methods of treating diseases therewith.

2. Background of the Invention

Many people suffer from inflammatory bowel disease (IBD). IBD is ageneric term used to refer to two inflammatory diseases, ulcerativecolitis and Crohn's disease, and various medications are being used totreat inflammatory bowel disease. For example, mesalamine,5-aminosalicylic acid (5-ASA) is used to treat ulcerative colitis, butis associated with various side effects. It can be absorbed as it passesthrough the GI tract, which can adversely affect the amount ofmesalamine that reaches the lower GI tract, particularly the colon andrectum. Sulfasalazine has also been used, but is metabolized in the bodyto form mesalamine (5-aminosalicylic acid (5-ASA)) and sulfapyridine.Accordingly, sulfasalazine is associated with several adverse sideaffects, including nausea, vomiting, abdominal discomfort, maleinfertility, and headache. These adverse side effects are usuallyattributed to the activity of sulfapyridine in the GI tract, as well asthat absorbed into the system. Olsalazine has also been used to treatulcerative colitis, but is both relatively expensive to make andassociated with adverse side effects including diarrhea.

Efforts have been made to minimize the side effects associated withthese compounds, including efforts by the present inventors to providecompounds that, when reaching the gut mucosa, break down into one ormore active compounds useful for treating inflammatory bowel disorders.Examples of such compounds are described, for example, in U.S. Pat. No.6,583,128 to Ekwuribe et al.

It would be beneficial to provide additional synthetic methods forpreparing these compounds. The present invention provides such syntheticmethods.

SUMMARY OF THE INVENTION

Synthetic methods for preparing compounds of the following formula areprovided:

where R¹, R³, and R⁴ are independently hydrogen or C₁ to C₄ alkyl, andR² is:

where R⁵ is selected from the group consisting of hydrogen and C₁ to C₄alkyl, or

where R⁶, R⁷ and R⁸ are independently hydrogen or C₁ to C₄ alkyl, aswell as the esters or pharmaceutically acceptable salts of suchcompounds.

The compounds prepared by these methods can be included inpharmaceutical compositions, and used in methods of treatinginflammatory conditions.

The active pharmaceutical ingredient may further comprise a compound ofFormula III:

The first step of the synthesis typically starts with an anilinecompound, which is converted to a diazonium salt. In one embodiment, theaniline compound to be converted to the diazonium salt includes aprimary alcohol or aldehyde group, which can be oxidized to a carboxylicacid group following formation of the diazonium salt and coupling to adesired molecule.

In another embodiment, the aniline compound to be converted to thediazonium salt includes a nitrile or amide group, which can behydrolyzed to the carboxylic acid following formation of the diazoniumsalt and coupling to a desired molecule.

The second step of the synthesis involves forming a diazonium salt,which can be performed using known chemistry. The resulting diazoniumsalt is then coupled with a compound of the formula:

This coupling step typically provides predominantly a substitution atthe 5-position of the salicylate, or a para substitution. In theembodiments where the aniline includes a primary alcohol or aldehydegroup, the next step involves oxidizing the primary alcohol or aldehydegroup to a carboxylic acid group. This can be accomplished usingtraditional oxidation chemistry, but optionally is performed using acatalytic amount of a chromium (VI) oxidant or other suitable oxidant inthe presence of a stoichiometric amount of periodic acid.

In the embodiments where the aniline includes a nitrile or amide group,the next step involves hydrolysis of the amide or nitrile to thecarboxylic acid. The hydrolysis can be either acid or base catalyzed.However, nitrilases and/or amidases can also be used, which typicallyresults in milder reaction conditions.

In either embodiment, following the oxidation of the primary alcohol oraldehyde to the carboxylic acid, or hydrolysis of the amide or nitrileto the carboxylic acid, a pharmaceutically acceptable carboxylate saltor suitable ester (i.e., where R¹ on the CO₂R¹ group is alkyl) can beformed using routine chemistry.

In an alternative embodiment of the first approach (where the aniline tobe converted to the diazonium salt includes a primary alcohol oraldehyde group, the methods first involve converting a compound of theformula:

to a compound of formula:

This conversion can be performed using standard reaction conditions wellknown to those of skill in the art, for example, as shown below withrespect to the nitrobenzene including the primary alcohol group:

In an alternative embodiment of the second approach (where the anilineto be converted to the diazonium salt includes a nitrile or amidegroup), the methods first involve converting a compound of the formulas:

to a compound of the formulas:

The amide group can optionally include one or two alkyl, aryl, arylalkylor alkylaryl groups in place of one or both of the hydrogen atoms, whichgroups are removed when the amide is hydrolyzed to the carboxylic acid.However, since such groups would not be present in the final molecule,it is often easier to simply use an unsubstituted amide group (i.e.,CONH₂).

This conversion can be performed using standard reaction conditions wellknown to those of skill in the art, for example, as shown below:

In still another embodiment, the starting materials for the reaction arenitrobenzene compounds such as:

which are coupled with a compound of the formula:

using a reagent such as lithium aluminum hydride (“LiAlH₄”), which isknown in the art to couple nitrobenzenes to form azo compounds. Desiredcompounds are isolated and purified.

Alternatively, two compounds of the formula:

can be coupled directly, forming an azo linkage. Potential limitationsto this approach are that when two different starting nitrobenzenes areused, three types of azo couplings are possible (A-A, A-B, B-B).Further, the reducing agent can reduce aldehyde, amide and nitrilegroups, which then need to be reoxidized to form the carboxylic acidmoieties. For this reason, it can be advantageous to perform the directazo coupling of nitrobenzenes with starting materials that alreadyinclude a carboxylic acid moiety (or carboxylate salt form thereof).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates embodiments of the synthesis routes described herein.

FIG. 2 illustrates additional embodiments of the synthesis routesdescribed herein.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described with respect to preferredembodiments described herein. It should be appreciated however thatthese embodiments are for the purpose of illustrating the invention, andare not to be construed as limiting the scope of the invention asdefined by the claims.

As shown below, various synthetic methods are provided for preparing thecompounds described herein.

In a first embodiment (Scheme I in FIG. 1), an aniline derivativeincluding a primary alcohol or aldehyde group is used, where the primaryalcohol or aldehyde group is ultimately oxidized to form a carboxylicacid. The carboxylic acid group can optionally be converted to apharmaceutically acceptable carboxylate salt or ester.

In a second embodiment (Scheme II in FIG. 2), an aniline derivativeincluding a nitrile or amide group is used, where the nitrile or amidegroup is ultimately hydrolyzed to form a carboxylic acid. The first stepin either embodiment involves forming a diazonium salt.

In a third embodiment, nitrobenzenes (rather than anilines) are used asstarting materials, and are reduced to form the aniline startingmaterials used in the first two embodiments.

In a fourth embodiment, two nitrobenzenes are directly coupled to forman azo linkage, using a reducing agent such as lithium aluminum hydride.

Each of these reaction conditions and the associated starting materialswill be better understood with reference to the following detaileddescription.

The Compounds

The compounds synthesized using the methods described herein includethose having the following formula:

where R¹, R³, and R⁴ are independently hydrogen or C₁ to C₄ alkyl.Preferably, R¹, R³, and R⁴ are independently selected from the groupconsisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂. More preferably, R¹, R³, andR⁴ are H or CH₃.

R² is:

R⁵ is selected from the group consisting of hydrogen and C₁ to C₄ alkyl.Preferably, R⁵ is selected from the group consisting of H, CH₃, CH₂CH₃,and CH(CH₃)₂. More preferably, R⁵ is H or CH₃ and, most preferably, R⁵is H. R⁶, R⁷ and R⁸ are independently hydrogen or C₁ to C₄ alkyl.Preferably, R⁶, R⁷ and R⁸ are independently selected from the groupconsisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂. More preferably, R⁶, R⁷ andR⁸ are independently H or CH₃.

Synthetic Methods

The individual reaction steps are discussed in more detail below.

Nitrobenzene Reduction to Aniline Starting Materials

In an alternative embodiment of the embodiment shown in Scheme 1 (wherethe aniline to be converted to the diazonium salt includes a primaryalcohol or aldehyde group), the methods first involve converting acompound of the formula:

to a compound of formula:

This conversion can be performed using standard reaction conditions wellknown to those of skill in the art, for example, as shown below:

In an alternative embodiment of the embodiment in Scheme II (where theaniline to be converted to the diazonium salt includes a nitrile oramide group), the methods first involve converting a compound of theformulas:

to a compound of the formulas:

where R¹ is as defined above. The amide group can include one or twoalkyl, aryl, arylalkyl or alkylaryl groups (in addition to those definedby R¹) such as are well known in the art in place of one or both of thehydrogen atoms, which groups are removed when the amide is hydrolyzed tothe carboxylic acid. However, since such groups would not be present inthe final molecule, it is often easier to simply use an unsubstitutedamide group (i.e., CONH₂).

This conversion can be performed using standard reaction conditions wellknown to those of skill in the art, for example, as shown below:

Conversion of Anilines to Diazonium Salts

The first step in Scheme I involves converting an aniline compound to adiazonium salt. The aniline compounds include a primary alcohol oraldehyde group, and the conversion involves the known reaction withNaNO₂ to convert anilines to diazonium salts. Such reaction conditionsare well known to those of skill in the art, and the reactions are shownbelow.

The first step in Scheme II also involves converting an aniline compoundto a diazonium salt. The aniline compounds include a nitrile or amidegroup, and the conversion similarly involves the known reaction withNaNO₂ to convert anilines to diazonium salts. The reactions are shownbelow.

In the reactions shown above, R¹ is as defined elsewhere in thisapplication. It is also contemplated that with respect to the amidegroups, any substitution on the amide linkage that does not interferewith the subsequent chemistry is acceptable, since the amide is laterhydrolyzed to the carboxylic acid and such groups will not appear, inany case, in the final molecule. Such groups include, for example, C₁₋₁₀straight, branched or cyclic alkyl groups, aryl groups such as thoseincluding one or more benzene or naphthalene rings, alkyl groupssubstituted with aryl groups (alkylaryl groups), aryl groups substitutedwith one to five alkyl groups (arylalkyl groups) and the like.

Electrophilic Aromatic Substitution Using Diazonium Salts

Electrophilic aromatic substitution reactions using diazonium salts arewell known. The following two aromatic compounds are substituted withthe previously described diazonium salts:

With respect to the first compound, substitution primarily takes placeat a position para to the phenol group and meta to the carboxylic acidgroup. With respect to the second compound, substitution primarily takesplace at a position para to the substituted (with R¹) acetic acid group.The following reactions take place in connection with Scheme I.

The following reactions take place with respect to Scheme II:

Oxidation of Primary Alcohol and Aldehyde Groups

With respect to Scheme I, the primary alcohol or aldehyde groups in theintermediate resulting from the diazonium coupling reaction are thenoxidized to carboxylic acid groups.

The reactions are shown below:

In some embodiments, particularly those embodiments where there is aphenol group present, the oxidation chemistry may oxidize the phenol toa quinone-type compound. In such embodiments, the phenol can beprotected with conventional protecting group chemistry (e.g., as a silylether, a THP ether, a benzyloxy, an ester, and the like), anddeprotected after the oxidation step. Examples of suitable protectinggroups for phenol groups which are amenable to various oxidationconditions are well known to those of skill in the art ((see Greene, T.W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.;Wiley: New York, 1991, the contents of which are hereby incorporated byreference).

U.S. Pat. No. 6,150,554 to Li et al., the contents of which are herebyincorporated by reference, teaches the oxidation of primary alcohols tocarboxylic acids using catalytic CrO₃ and periodic acid (referred to inthe literature as both HIO₄ and H₅IO₆) as the stoichiometric oxidant.This chromium catalyzed oxidation method avoids the chromium disposalissues associated with running a typical Jones oxidation reaction withstoichiometric CrO₃, reduces the epimerization of any α-chiral centers,and is a one step procedure. The reaction is mild, rapid, high yieldingand only requires 1-2 mol % of CrO₃.

The oxidation of primary alcohols to carboxylic acids is well known inthe art of organic synthesis. Typical reagents include chromium (VI)oxidizing agents (including, but not limited to, sodium and potassiumdichromate, chromic acid, and the like), and manganese (VII) oxidizingagents such as potassium permanganate. The oxidation of aldehydes tocarboxylic acids is also well known. While the same strong oxidizingagents used to oxidize primary alcohols can be used, much milderoxidizing agents can also be used. For example, ozone (or oxygen) andultraviolet light and/or a suitable catalyst can often be used.Representative reaction conditions are described, for example, in U.S.Pat. No. 6,680,395 to Springer, the contents of which are herebyincorporated by reference.

Industrially, it is often preferred to use the mildest and/or leastexpensive oxidation conditions that result in the desired products.Accordingly, it can be preferred to use oxygen or ozone as an oxidizingagent where appropriate, or to use catalytic chromium (VI) salts alongwith a co-oxidizing agent such as periodic acid to regenerate thecatalytic chromium (VI) salts where appropriate.

Hydrolysis of Nitrile and Amide Groups

The embodiment in Scheme II does not require the use of primary alcoholsin the starting material, so that oxidation from a primary alcohol to acarboxylic acid group can be avoided. This embodiment involves the useof an aniline that includes a nitrile (—CN) or amide (—C(O)NR¹ ₂) groupin place of the primary alcohol group. The nitrile or amide groups canbe hydrolyzed to a carboxylic acid group after the diazonium salt iscoupled to a desired aromatic ring. The reactions are shown below.

Reaction conditions for converting a nitrile to a carboxylic acid groupare well known to those of skill in the art, and are described, forexample, in March, J. Advanced Organic Chemistry; 3rd ed., John Wiley:New York, (1985) and House, Modern Synthetic Reactions, 2d. ed., W. A.Benjamin, Inc., Menlo Park, Calif., (1972). The conditions involveeither using an acid or a base to catalyze the hydrolysis. A tautomer ofan amide is believed to be an intermediate in the hydrolysis of anitrile group to a carboxylic acid. For this reason, an amide group canbe used in place of the nitrile for purposes of carrying out thesynthetic method.

Biocatalytic hydrolysis of nitriles to carboxylic acids can be carriedout under mild conditions, without affecting other functional groups.The nitrile group thus functions as a “masked” acid group. Nitrilaseenzymes (for example, those from Arabidopsis thaliana, Alcaligenesfaecalis, Pseudomonas fluorescens, Rhodococcus rhodochrous, andRhodococcus sp.) are available commercially from companies such asSigma-Adrich. The chemistry is described, for example, in Effenger andOsswald, “Catalyst for the (E)-selective hydrolysis of(E,Z)-α,β-unsaturated nitriles to carboxylic acids,” Tetrahedron:Asymmetry, 12,:2581 (2001); Effenger and Osswald, “Selective hydrolysisof aliphatic dinitriles to monocarboxylic acids, Synthesis, 1866 (2001);and Effenger and Osswald, “Enantioselective hydrolysis of(1)-arylacetonitriles, Tetrahedron: Asymmetry, 12:279 (2001), thecontents of which are hereby incorporated by reference. Because thebiocatalytic hydrolysis can involve milder conditions than either theacid or basic hydrolysis of nitriles, this method can be preferred,particularly for industrial synthesis of the compounds described herein.

A large number of amidases (enzymes that cleave amide groups) are alsoknown and can be used to hydrolyze the amides to carboxylic acids. Onepotential advantage of using the amidases is that they tend to beselective for one enantiomer over another. In some embodiments, thecompounds are formed as racemic mixtures or mixtures of diastereomers,and the amidases allow (via enzymatic resolution) the individualenantiomers or diastereomers to be separated. Of course, conventionalmethods, such as selective crystallization, chiral chromatography andthe like, as are well known to those of skill in the art, can also beused to isolate enantiomerically enriched compounds.

The resulting carboxylic acid-containing compounds can often be easilyseparated from the amide-containing compounds by simply forming thecarboxylate salts, which can be removed in a water extraction and thenisolated by acidification of the carboxylate group to reform thecarboxylic acid group. The amide groups not hydrolyzed by the enzyme(or, for that matter, via conventional acidic or basic hydrolysis) canbe isolated from extraction with an organic solvent, and subjected toalternate hydrolysis conditions.

Optionally, the carboxylic acid groups can be converted topharmaceutically acceptable carboxylate salts or esters, using knownreaction conditions.

Direct Azo Coupling of Nitrobenzenes Using Reducing Agents

Nitrobenzene starting materials can be directly coupled to form azolinkages using a reducing agent such as lithium aluminum hydride.Representative reactions using the nitrobenzene compounds are shownbelow:

In the first instance, the three different azo coupling products willlikely have to be separated. In the second instance, the compounds areeither identical (X is CO₂H or CO₂ ⁻), or X is readily convertible to acarboxylic acid group, so that separation need not be performed. Thatis, if X is an aldehyde or primary alcohol, it can be oxidized in thepresence of the carboxylic acid group to yield the desired compound. IfX is a nitrile or amide group, it can be hydrolyzed in the presence ofthe carboxylic acid group to yield the desired compound. For thesereasons, it may be advantageous to use this embodiment (azo coupling ofnitrobenzenes) only when the molecules are either symmetrical or wouldbe symmetrical when X is converted to a carboxylic acid group. It isalso worth noting that aldehyde, amide and nitrile groups can be reducedwith the reducing agent (for example, lithium aluminum hydride). Thestoichiometric equivalents of the reducing agent may need to be adjustedto account for this, and any reduced groups will need to be oxidized toform the carboxylic acid. For this reason, it may be preferred to usecompounds where X is a carboxylic acid or carboxylate group whenperforming this reaction. Finally, any suitable carboxylate salt can beused (that is, any metal cation or ammonium cation can be used) if thedesired end product includes a carboxylic acid at that position, as theacidification of virtually any carboxylate salt to form the carboxylicacid is routine in the art. That is to say, for cost reasons, it may bepreferred to use a sodium or potassium salt, although other salts wouldcertainly be acceptable.

Pharmaceutical Formulations

The compounds prepared according to the methods described herein can beincluded in pharmaceutical formulations, both for veterinary and forhuman medical use. The compounds can be administered per se as well asin the form of pharmaceutically acceptable esters, salts, and otherphysiologically functional derivatives thereof.

The formulations include those suitable for parenteral as well asnon-parenteral administration, and specific administration modalitiesinclude, but are not limited to, oral, rectal, buccal, topical, nasal,ophthalmic, subcutaneous, intramuscular, intravenous, transdermal,intrathecal, intra-articular, intra-arterial, sub-arachnoid, bronchial,lymphatic, vaginal, and intra-uterine administration. Formulationssuitable for oral and parenteral administration are preferred, withformulations suitable for oral administration most preferred.

In addition to the aforementioned ingredients, the formulations of thisinvention can further include one or more accessory ingredient(s)selected from diluents, buffers, flavoring agents, disintegrants,surface active agents, thickeners, lubricants, preservatives (includingantioxidants), and the like.

The active pharmaceutical ingredient can further include one or moreother medicaments including, but not limited to, anti-inflammatoryagents such as mesalamine, sulfasalazine, balsalazide, and olsalazine;immunomodulators such as azathioprine, 6-mercaptopurine, cyclosporineand methotrexate; steroidal compounds such as corticosteroids; andantibiotics such as metronidazole and ciprofloxacin. The activepharmaceutical ingredient preferably further comprises mesalamine, thecompound of Formula II:

In therapeutic usage, the compounds can be used to treat an animalsubject having or latently susceptible to an intestinal condition(s) ordisease state(s) and in need of treatment therefor, by administering aneffective amount of the compound to the animal. The animal can be ahuman or non-human animal (e.g., bird, dog, cat, cow, horse), preferablya mammal, and most preferably a human.

The compounds can be used to treat and/or prevent various diseases,particularly inflammatory conditions of the GI tract including, but notlimited to, inflammatory conditions of the mouth such as mucositis,infectious diseases (e.g., viral, bacterial, and fungal diseases), andCrohn's disease; inflammatory conditions of the esophagus such asesophagitis, conditions resulting from chemical injury (e.g., lyeingestion), gastroesophageal reflux disease, bile acid reflux, Barrett'sesophagus, Crohn's disease, and esophageal stricture; inflammatoryconditions of the stomach such as gastritis (e.g., Helicobacter pylori,acid-peptic disease and atrophic gastritis), peptic ulcer disease,pre-cancerous lesions of the stomach, non-ulcer dyspepsia, and Crohn'sdisease; inflammatory conditions of the intestine such as inflammatorybowel disease, celiac disease, Crohn's disease, bacterial overgrowth,peptic ulcer disease, and fissures of the intestine; inflammatoryconditions of the colon such as Crohn's disease, ulcerative colitis,infectious colitis (e.g., pseudomembranous colitis such as Clostridiumdifficile colitis, salmonella enteritis, shigella infections,yersiniosis, cryptosporidiosis, microsporidial infections, and viralinfections), radiation-induced colitis, colitis in the immunocompromisedhost (e.g., typhlitis), precancerous conditions of the colon (e.g.,dysplasia, inflammatory conditions of the bowel, and colonic polyps),proctitis, inflammation associated with hemorrhoids, proctalgia fugax,and rectal fissures; liver gallbladder and/or bilary tract conditionssuch as cholangitis, sclerosing cholangitis, primary bilary cirrhosis,and cholecystitis; and intestinal abscess. The compounds can also beused to diagnose constituents, conditions, or disease states inbiological systems or specimens, as well as for diagnostic purposes innon-physiological systems. Furthermore, the compounds can be used totreat or prevent condition(s) or disease state(s) in plant systems. Byway of example, the compounds or individual active components of theconjugate can have insecticidal, herbicidal, fungicidal, and/orpesticidal efficacy amenable to usage in various plant systems.

Depending on the specific condition or disease state, the activepharmaceutical ingredient can be administered at any suitabletherapeutically effective and safe dosage, as can readily be determinedwithin the skill of the art and without undue experimentation. Forexample, the active pharmaceutical ingredient of the present inventioncan be administered at a dosage between about 0.1 and 200 mg/kg,preferably between about 1 and 90 mg/kg, and more preferably betweenabout 10 and 80 mg/kg.

In some embodiments, the compounds can break down in the intestinaltract to form the metabolic product of Formula III:

where R¹, R³ and R⁴ are as described above with reference to Formula I,and the metabolic product of Formula IV:

The metabolic product of Formula III can possess anti-inflammatoryactivity and/or immunoregulatory activity. The metabolic product ofFormula IV can possess anti-inflammatory activity, and more particularlycan inhibit prostaglandin synthetase I & II.

In other embodiments, the compounds can break down in the intestinaltract to form the metabolic product of Formula III and the metabolicproduct of Formula V:

where R⁶, R⁷ and R⁸ are as described above with reference to Formula I.The metabolic product of Formula V can possess anti-inflammatoryactivity and/or immunoregulatory activity.

Accordingly, the compounds can provide immunoregulatory activity. Thecompounds can also inhibit prostaglandin synthetase I and II. Thecompounds can be used to treat various diseases, particularly ulcerativecolitis, Crohn's disease and the like.

The present invention will now be described with reference to thefollowing examples. It should be appreciated that these examples are forthe purposes of illustrating aspects of the present invention, and donot limit the scope of the invention as defined by the claims.

EXAMPLES Example 1 Diazo Coupling of 4-Aminophenethyl Alcohol withSalicylic Acid, Followed by Oxidation of the Alcohol to theCorresponding Carboxylic Acid

Diazo Coupling of 4-Aminophenethyl Alcohol with Salicylic Acid:

4-Aminophenethyl alcohol (24.8 mmol) is suspended in water (75 mL) andconcentrated hydrochloric acid (8 mL) is added. The solution is cooledto 0° C. in an ice bath with rapid stirring. Sodium nitrite (26.1 mmol)in water (20 mL) is added dropwise to the 4-aminophenylacetic acidsolution with rapid stirring. It may be important to keep thetemperature between 0-5° C. at all times, especially during the NaNO₂addition. The reaction is stirred for an additional period of time, forexample, about 20 minutes. In the meantime, salicylic acid, sodium salt(74.4 mmol) is dissolved in an aqueous NaOH solution (113 mmol NaOH in100 mL H₂O). The solution is vigorously stirred at 17° C. and at pH13.3. The diazonium salt solution is added dropwise to the salicylicacid solution. It can be important to keep the temperature of thesalicylic acid solution between 17-18° C. and the pH between 13.2-13.3at all times, especially during the diazonium salt addition. Thetemperature can be regulated by adding ice and the pH regulated byadding NaOH, for example, 8M NaOH. After the addition is complete, thesolution is allowed to warm room temperature and stirred for anadditional period of time, for example, about 30 minutes. The reactionmixture is then suction filtered to remove any undissolved particulatesor unwanted side products. The filtrate is acidified with aqueous HCl(10 mL conc. HCl in 20 mL H₂O) which produces a dark red precipitate.The precipitate is collected by suction filtration and washed severaltimes with cold H₂O, until the filtrate is clear. The collected solidcan be air dried overnight.

Oxidation of the Resulting Alcohol to the Corresponding Carboxylic Acid:

A mixture of alcohol formed above (0.7 mol) and a solution of sodiumcarbonate is placed in 150 mL of water. A solution of potassiumpermanganate (0.9 mol) in water is added, with vigorous stirring, for3-4 hours at a temperature of 4-5° C. The reaction mixture is allowed towarm to room temperature and the precipitated manganese dioxide isremoved by suction filtration. The solution is concentrated underreduced pressure, a layer of ether is added, and the aqueous solution isacidified with dilute H₂SO₄. The organic and aqueous phases areseparated and the aqueous phase extracted 2-3 times with ether. Theether extracts are combined, dried over MgSO₄, and the solvent removedunder reduced pressure. The resulting solid can optionally berecrystallized.

Example 2 Diazo Coupling of 4-Aminobenzyl Cyanide with Salicylic Acid,Followed by Hydrolysis of the Nitrile to the Corresponding CarboxylicAcid

Diazo Coupling of 4-Aminobenzyl Cyanide with Salicylic Acid:

4-Aminobenzyl cyanide (24.8 mmol) is suspended in water (75 mL) andconcentrated hydrochloric acid (8 mL) is added. The solution is cooledto 0° C. in an ice bath with rapid stirring. Sodium nitrite (26.1 mmol)in water (20 mL) is added dropwise to the 4-aminophenylacetic acidsolution with rapid stirring. It is preferred to keep the temperaturebetween 0-5° C. at all times, especially during the NaNO₂ addition. Thereaction is stirred for an additional 20 minutes. In the meantime,salicylic acid, sodium salt (74.4 mmol) is dissolved in an aqueous NaOHsolution (113 mmol NaOH in 100 mL H₂O). The solution is vigorouslystirred at 17° C. and at pH 13.3. The diazonium salt solution is addeddropwise to the salicylic acid solution. It can be extremely importantto keep the temperature of the salicylic acid solution between 17-18° C.and the pH between 13.2-13.3 at all times, especially during thediazonium salt addition. The temperature is regulated by adding ice andthe pH regulated by adding NaOH, for example, 8M NaOH. After theaddition is complete, the solution is allowed to warm room temperatureand stirred for an additional period of time, such as about 30 minutes.The reaction mixture can be suction filtered to remove any undissolvedparticulates or unwanted side products, and the filtrate can beacidified with aqueous HCl (10 mL conc. HCl in 20 mL H₂O) to produce adark red precipitate. The precipitate can be collected by suctionfiltration and washed several times with cold H₂O, until the filtrate isclear. The collected solid can be air dried overnight.

Hydrolysis of the Nitrile to the Corresponding Carboxylic Acid a) BasicConditions:

The nitrile formed above (1.2 mol) and a solution of NaOH (2.3 mol) canbe placed in H₂O (260 mL) in a round-bottom flask and heat to refluxusing a condenser for approximately 5-10 h. Water (100 mL) can be addedthrough the condenser, then slowly with external cooling, 50% H₂SO₄ canbe added. The upper layer containing the desired carboxylic acid can beseparated and extracted with CH₂Cl₂. The organic extracts can becombined, dried over MgSO₄, and the solvent removed under reducedpressure. The resulting solid can optionally be recrystallized.

b) Acidic Conditions:

The nitrile formed above (0.85 mol) and a solution of 50:50H₂SO₄:glacial acetic acid can be placed in H₂O (100 mL) in around-bottom flask and heated to reflux using a condenser forapproximately 1 hour. The reaction mixture can be allowed to cool toroom temperature and poured into 2-3 volumes of water with stirring. Thedesired crude product should precipitate. The resulting solid can becollected by suction filtration and washed with water until the filtrateis neutral. The resulting solid can optionally be recrystallized.

Example 3 Reduction of Nitro Compounds to Form an Azo Linkage

In this embodiment, two nitrobenzene compounds (shown below as ArNO₂)are coupled to form a direct azo linkage between the two compounds. Thisembodiment will result in mixtures of products (A-A, A-B and B-B) if twodifferent nitrobenzene compounds are used. For this reason, it may bepreferably to limit this synthetic approach to the synthesis ofsymmetrical azo compounds, e.g. 4-APAA dimer, etc. (Ar=Phenyl ring)

A solution of NaOH (1.6 mol) in H₂O (150 mL), nitrobenzene (0.41 mol)and MeOH (500 mL) is placed in a 3-neck flask equipped with an overheadstirrer and reflux condenser. Zn powder (0.9 mol) is added and refluxedwith vigorous stirring for approximately 10 h. The reaction mixture ishot filtered and the precipitate washed with MeOH. Concentrated HCl isadded to the filtrate until the pH is neutral by litmus paper, and theprecipitated is re-filtered. The MeOH is removed under reduced pressureand the resulting aqueous solution is cooled in an ice bath until thedesired azo compound precipitates. The solid can then be collected bysuction filtration and optionally recrystallized.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A method for preparing a compound of the formula:

where R¹, R³ and R⁴ are is selected from the group consisting of H, CH₃,CH₂CH₃, and CH(CH₃)₂; and R² is:

where R⁵, R⁶, R⁷ and R⁸ are independently hydrogen or C₁ to C₄ alkyl; orthe esters or pharmaceutically acceptable salts thereof, comprising: a)combining a first compound of formula

where X is selected from the group consisting of CH₂OH, C(O)H, CO₂H, CO₂⁻, CN, and C(O)NR¹ ₂, with a second compound of formula

b) adding a reducing agent that is capable of reducing the nitro groupsin the first and second compounds and forming an azo group, and c)converting any X groups that are not in the form of carboxylic acid,carboxylate salt or ester groups to such groups.
 2. The method of claim1, wherein R¹, R³, and R⁴ are independently selected from the groupconsisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂.
 3. The method of claim 1,wherein R⁶, R⁷, and R⁸ are independently selected from the groupconsisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂.
 4. The method of claim 1,wherein R⁵ is selected from the group consisting of H, CH₃, CH₂CH₃, andCH(CH₃)₂.
 5. The method of claim 1, wherein the compound that is formedis 5-(4-carboxymethyl-phenylazo)-2-hydroxy-benzoic acid.
 6. The methodof claim 1, wherein the compound that is formed is5-[4-(1-carboxy-ethyl)-phenylazo]-2-hydroxy-benzoic acid.
 7. The methodof claim 1, wherein the compound that is formed is4-(4-carboxymethyl-phenylazo)-phenylacetic acid.
 8. The method of claim1, further comprising removing products formed by the coupling of two ofthe first compounds and/or two of the second compounds from the desiredproduct resulting from the coupling of the first and second compound. 9.The method of claim 1, wherein X is CN or C(O)NR¹ ₂, wherein the CN orC(O)NR¹ ₂ groups are either hydrolyzed to form carboxylic acids orcarboxylate salts, or, in the event that the CN or C(O)NR¹ ₂ groups arereduced by the reducing agent during the coupling of the nitro groups,such reduced groups are converted to carboxylic acid groups orcarboxylate salts.
 10. The method of claim 9, wherein one or more of thecarboxylic acids or carboxylate salts are esterified.
 11. The method ofclaim 1, wherein X is CH₂OH or C(O)H, further comprising oxidizing theCH₂OH or C(O)H groups to form a carboxylic acid.
 12. The method of claim1, wherein the oxidation of the primary alcohol or aldehyde group isperformed using a chromium (VI) oxidizing agent.
 13. The method of claim12, wherein the chromium (VI) oxidizing agent is present in catalyticamounts, and a co-oxidant is used to regenerate the chromium (VI)oxidizing agent.
 14. The method of claim 1, wherein the compound existsas a mixture of diastereomers, further comprising isolating anindividual enantiomer from the mixture of diastereomers.